Summary Defects in motor neuron (MN) function or survival result in severe human pathologies, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), with distinct MN subtypes differing in their susceptibility to disease. There is currently no effective treatment for MN disorders in part due to a lack of understanding of the molecular mechanisms that allow distinct MN subtypes to acquire and maintain their function-defining properties. Thus, basic research in model organisms such as nematodes, flies, and mice is needed to reveal such mechanisms. The underlying basis of MN subtype function is the differential expression of MN subtype-specific terminal differentiation genes, which encode proteins (e.g. ion channels, neurotransmitter receptors) that define the functional properties of a given MN subtype throughout life. Here, we propose a novel approach that specifically focuses on the transcriptional regulation of MN subtype-specific terminal differentiation genes. Our goal is to uncover the gene regulatory mechanisms that establish during development and maintain throughout life the expression of MN subtype-specific terminal differentiation genes. Such knowledge will advance our understanding of how MN subtype-specific functional properties are established and maintained, thereby providing new insights into the etiology and diagnosis of MN disorders. The availability of MN subtype-specific terminal differentiation markers in C. elegans has enabled us to identify three highly conserved gene regulatory factors, the transcription factors UNC-3 and CFI-1 and the chromatin remodeling factor PBRM-1, that determine MN subtype function by regulating the expression of terminal differentiation genes. Intriguingly, our results suggest that while UNC-3 activates expression of MN subtype-specific terminal differentiation genes, the regulatory factors CFI-1 and PBRM-1, counteract this activator function by repressing UNC-3 targets in specific MN subtypes. These observations are important because they could be indicative of a general mechanism for the acquisition and maintenance of MN subtype- specific features in which the transcriptional targets of a broadly acting activator (UNC-3) are repressed in a MN subtype-specific fashion (by CFI-1 and PBRM-1). To test this idea within the 2-year R21 timeframe, we will employ an innovative approach that focuses on UNC-3 and CFI-1. First, we have devised a new strategy to isolate distinct C. elegans MN subtypes, thereby enabling the identification of UNC-3 and CFI-1 transcriptional targets through RNA-sequencing (Aim 1). Second, we will employ the powerful auxin-inducible degradation (AID) system to test the hypothesis that UNC-3 and CFI-1, apart from their developmental function, also exhibit a maintenance role by continuously controlling the expression of MN subtype-specific terminal differentiation genes throughout life (Aim 2). Our findings may serve as a blueprint for future investigations of gene regulatory mechanisms for neuronal subtype development and function throughout the nervous system.